The Use of PRV-Bartha to Define Premotor Inputs to Lumbar Motoneurons in the Neonatal Spinal Cord of the Mouse

The neonatal mouse has become a model system for studying the locomotor function of the lumbar spinal cord. However, information about the synaptic connectivity within the governing neural network remains scarce. A neurotropic pseudorabies virus (PRV) Bartha has been used to map neuronal connectivity in other parts of the nervous system, due to its ability to travel trans-neuronally. Its use in spinal circuits regulating locomotion has been limited and no study has defined the time course of labelling for neurons known to project monosynaptically to motoneurons.Here we investigated the ability of PRV Bartha, expressing green and/or red fluorescence, to label spinal neurons projecting monosynaptically to motoneurons of two principal hindlimb muscles, the tibialis anterior (TA) and gastrocnemius (GC). As revealed by combined immunocytochemistry and confocal microscopy, 24-32 h after the viral muscle injection the label was restricted to the motoneuron pool while at 32-40 h the fluorescence was seen in interneurons throughout the medial and lateral ventral grey matter. Two classes of ipsilateral interneurons known to project monosynaptically to motoneurons (Renshaw cells and cells of origin of C-terminals) were consistently labeled at 40 h post-injection but also a group in the ventral grey matter contralaterally. Our results suggest that the labeling of last order interneurons occurred 8-12 h after motoneuron labeling and we presume this is the time taken by the virus to cross one synapse, to travel retrogradely and to replicate in the labeled cells.The study establishes the time window for virally-labelling monosynaptic projections to lumbar motoneurons following viral injection into hindlimb muscles. Moreover, it provides a good foundation for intracellular targeting of the labeled neurons in future physiological studies and better understanding the functional organization of the lumbar neural networks

A cluster of cholinergic premotor interneurons modulates mouse locomotor activity

Mammalian motor programs are controlled by networks of spinal interneurons that set the rhythm and intensity of motor neuron firing. Motor neurons have long been known to receive prominent “C bouton” cholinergic inputs from spinal interneurons, but the source and function of these synaptic inputs have remained obscure. We show here that the transcription factor Pitx2 marks a small cluster of spinal cholinergic interneurons, V0C neurons, that represents the sole source of C bouton inputs to motor neurons. The activity of these cholinergic interneurons is tightly phase locked with motor neuron bursting during fictive locomotor activity, suggesting a role in the modulation of motor neuron firing frequency. Genetic inactivation of the output of these neurons impairs a locomotor task-dependent increase in motor neuron firing and muscle activation. Thus, V0C interneurons represent a defined class of spinal cholinergic interneurons with an intrinsic neuromodulatory role in the control of locomotor behavior.Publisher PDFPeer reviewe

Genetic Inactivation of Cholinergic C Bouton Output Improves Motor Performance but not Survival in a Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects upper and lower motor neurons and leads to death a few years after symptom onset. Despite its high morbidity and mortality, its underlying pathogenic mechanisms still remain poorly understood. Although there is increasing evidence for significant changes in the structure and function of synapses on motor neurons, there is a need for a systematic investigation of the role of each synapse subtype in the course of the disease. Here, we focus on large cholinergic synapses on motor neurons, known as C boutons, and investigate their role during ALS progression. We implement a genetic strategy for inactivation of the cholinergic output of C boutons in the SOD1G93A transgenic mouse model of ALS. We demonstrate that although C bouton cholinergic inactivation does not alter mouse survival, it exerts a beneficial effect on motor performance in the rotarod motor task, as evidenced by an increased latency to fall in SOD1G93A mice lacking C bouton cholinergic output. Our results suggest that C bouton cholinergic transmission exerts a negative effect on motor neuron function in ALS, possibly via aberrant excitation, and render C boutons a potential target for future pharmacological intervention. © 2020 IBR

A cluster of cholinergic premotor interneurons modulates mouse locomotor activity

Mammalian motor programs are controlled by networks of spinal interneurons that set the rhythm and intensity of motor neuron firing. Motor neurons have long been known to receive prominent “C bouton” cholinergic inputs from spinal interneurons, but the source and function of these synaptic inputs have remained obscure. We show here that the transcription factor Pitx2 marks a small cluster of spinal cholinergic interneurons, V0C neurons, that represents the sole source of C bouton inputs to motor neurons. The activity of these cholinergic interneurons is tightly phase locked with motor neuron bursting during fictive locomotor activity, suggesting a role in the modulation of motor neuron firing frequency. Genetic inactivation of the output of these neurons impairs a locomotor task-dependent increase in motor neuron firing and muscle activation. Thus, V0C interneurons represent a defined class of spinal cholinergic interneurons with an intrinsic neuromodulatory role in the control of locomotor behavior

Germ line transformation of the olive fly Bactrocera oleae using a versatile transgenesis marker. Insect Molecular Biology 15, 95103. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to be placed on the EU Market The presen

Abstract The olive fruit fly (olive fly) Bactrocera oleae (Dacus), recently introduced in North America, is the most destructive pest of olives worldwide. The lack of an efficient gene transfer technology for olive fly has hampered molecular analysis, as well as development of genetic techniques for its control. We have developed a Minos -based transposon vector carrying a selfactivating cassette which overexpresses the enhanced green fluorescent protein (EGFP). Efficient transposasemediated integration of one to multiple copies of this vector was achieved in the germ line of B. oleae by coinjecting the vector along with in vitro synthesized Minos transposase mRNA into preblastoderm embryos. The self-activating gene construct combined with transposase mRNA present a system with potential for transgenesis of very diverse species

A cluster of cholinergic premotor interneurons modulates mouse locomotor activity

SummaryMammalian motor programs are controlled by networks of spinal interneurons that set the rhythm and intensity of motor neuron firing. Motor neurons have long been known to receive prominent “C bouton” cholinergic inputs from spinal interneurons, but the source and function of these synaptic inputs have remained obscure. We show here that the transcription factor Pitx2 marks a small cluster of spinal cholinergic interneurons, V0C neurons, that represents the sole source of C bouton inputs to motor neurons. The activity of these cholinergic interneurons is tightly phase locked with motor neuron bursting during fictive locomotor activity, suggesting a role in the modulation of motor neuron firing frequency. Genetic inactivation of the output of these neurons impairs a locomotor task-dependent increase in motor neuron firing and muscle activation. Thus, V0C interneurons represent a defined class of spinal cholinergic interneurons with an intrinsic neuromodulatory role in the control of locomotor behavior

Subcortical Visual Shell Nuclei Targeted by ipRGCs Develop from a Sox14+-GABAergic Progenitor and Require Sox14 to Regulate Daily Activity Rhythms

SummaryIntrinsically photosensitive retinal ganglion cells (ipRGCs) and their nuclear targets in the subcortical visual shell (SVS) are components of the non-image-forming visual system, which regulates important physiological processes, including photoentrainment of the circadian rhythm. While ipRGCs have been the subject of much recent research, less is known about their central targets and how they develop to support specific behavioral functions. We describe Sox14 as a marker to follow the ontogeny of the SVS and find that the complex forms from two narrow stripes of Dlx2-negative GABAergic progenitors in the early diencephalon through sequential waves of tangential migration. We characterize the requirement for Sox14 to orchestrate the correct distribution of neurons among the different nuclei of the network and describe how Sox14 expression is required both to ensure robustness in circadian entrainment and for masking of motor activity.Video Abstrac